1. Field of the Invention
The present invention relates to novel fractions extracted from aerobic bacteria, endowed with antitumoral, antibacterial and interferon inducing properties and to the process for their preparation.
2. Description of the Prior Art
Research investigations have established the immunostimulant and antitumoral power of killed microbial bodies of certain species of anaerobic Corynbacteriae [cf. notably A. R. PREVOT, B. N. HALPERN, F. BIOZZI, C. STIFFEL, D. MOUTON, J. C. MORARD, Y. BOUTHILLIER, C. DECREUSEFOND, C.R. Acad. Sci. (PARIS), 1963, 257 (Series D), 13]. Studies were then devoted to the isolation of active fractions from whole germs [cf. notably M. RAYNAUD, B. KOUZNETZOVA, B. BIZZINI and J. C. CHERMANN: "Study of the immunostimulant effect of various species of anaerobic Corynbacteriae and their fractions" Ann. Inst. PASTEUR, 1972, 122, 695-700; A. R. PREVOT, M. RAYNAUD, B. BIZZINI, J. C. CHERMANN, B. KOUZNETZOVA and F. SINOUSSI: "Reticulostimulant Activity of the walls of anaerobic Corynebacteriae "C.R. Acad. Sci. PARIS, t. 274 (Apr. 10, 1972)]; "B. KOUZNETZOVA and B. BIZZINI, J. C. CHERMANN, F. DEGRAND, A. PREVOT and M. RAYNAUD: "Recent results in cancer research" 1974, 47, 275-293 (Immunostimulating activity of whole cells, cellwalls and fractions of anaerobie corynebacteria)". D. MIGLIORE-SAMOUR and J. JOLLES [Febs Letters 25 no. 2 (1972) 301] have described the isolation from anaerobic Corynebacteriae, of a water-soluble fraction which has preserved the adjuvant power of the whole bacteria. Then P. LALLOUETTE, B. BIZZINI and M. RAYNAUD [N.G.M. 25(1975) 13], by applying the process of MIGLIORE-SAMOUR and JOLLES to the fractionation of Corynebacterium granulosum, found that the adjuvant and immunostimulant activity was associated not with the water-soluble fraction S.sub.70, but with the particulate fraction P.sub.40. The fraction P.sub.40 isolated from C. granulosum was characterized chemically and biologically by B. BIZZINI, B. MARO and P. LALLOUETTE, [in M/e/ decine et maladies infectieuses 78, no. 9, vol. VIII, pages 408-414 (isolation and characterisation of a so-called P.sub.40 fraction from Corynebacterium granulosum)]. They established the particularly interesting curative action of the P.sub.40 fraction on the P815 tumor in the mouse. On the other hand, P. LALLOUETTE, B. MARO, A. SCHWARTZ, B. BIZZINI (cf. C.R. Acad. Sci. (PARIS), 1976), showed that the P.sub.40 fraction is capable of restoring and of increasing the immunogenic capacity of the formation of sheep anti-red blood cell antibodies in mice depressed by the injection of cyclophosphamide.
There exist in addition, a certain number of studies regarding the obtaining of interferon inducers, and notably the French MAES Pat. No. 2,092,875, relating to complexes inducing the formation of interferon and to the method for their preparation. These complexes are obtained by reacting a mono-, bi- or tricatenary nucleic acid with a dialkylaminoalkyl dextran in a weight ratio alkylaminoalkyldextran/nucleic acid less than 1/1 or with a synthetic polymer of aminoacids such as polylysine or polyornithine or indeed with basic natural proteins such as histone or lysozyme, or again with a synthetic cationic polyelectrolyte such as hexadimethrine bromide. French Pat. No. 2,130,533 in the name of The Kotasato Institute also describes a slightly toxic interferon inducer devoid of side effects, obtained from the supernatant liquor of a liquid culture medium of an organism of phase III, of phase II or of phase I of Bordetella pertussis, or from a fraction obtained by disintegration of the protoplasm of an organism of phase III, of phase II or of phase I of Bordetella pertussis and subsequent separation of the lipopolysaccharide endotoxin and of the O-antigen of the somatic components of the bacteria. This patent sets out in addition the state of the art in prior publications [Julius S. YOUNGER, Journal General Physiology, 56 (1), Part 2, pages 25-40 (1970) and J. VILCEK, Virology Monograph, Vol. 6, Interferon, Editions SPRINGER, 1969, P. 21-22] which describe interferon inducers for the prevention of viral infectious diseases, such as various viruses, bacteria (in particular Gram negative bacteria), the lipopolysaccharide endotoxins of these bacteria, metabolic products of molds, double-helix polysaccharides and ribonucleic acids, all considered as toxic and inducing undesirable side actions. The UPJOHN American U.S. Pat. No. 4,007,086 describes, for its part, a process for interferon production in vitro which consists of subjecting human or animal interferon producing cells to ultra-violet radiation of 50 ergs/mm.sup.2 to 2,500 ergs/mm.sup.2, before, at the same time or after the addition to these cells of a non-viral interferon inducer. The interferon inducer applied according to this Patent may be an endotoxin, a bacterium, a trachomatous conjunctivity agent, a mycoplasm, a protozoan, a rickettsia, a synthetic polymer, a mitogen, a polysaccharide, an antibiotic, an interferon inducer of low molecular weight such as tilorone hydrochloride, natural and synthetic nucleic acids, and notably mono- or di-catenary ribonucleic acids and their complexes with polysaccharides and other substances.
The possibility of having availabe a non-toxic interferon inducer devoid of side effects is all the more important in that interferon is only produced in the presence of interferon inducers. Now, interferon, which is a protein which appears in the blood or the organs of animals or in tissue culture media when the latter are subjected to the action of an interferon inducer, prevents or attenuates viral diseases.
It is an object of the present invention to provide a novel agent having at the same time, adjuvant properties, immunostimulant properties, antibacterial properties, and interferon inducing properties.
As has been shown above, the adjuvant and immunostimulant power of anaerobic Corynebacteriae has been amply demonstrated. On the other hand, tests carried out on known aerobic Corynebacteriae have shown that the latter are devoid of adjuvant and immunostimulant properties (Comptes-rendus de l'Academie des Sciences Paris 1964, vol. 258, p. 4619-4621).
Applicants have isolated from the sputum of a patient afflicted with generalized cancer with extensive pulmonary and mesenteric metastases, a new Corynebacterium aerobe, in pure culture directly.
The culture of this Corynebacterium aerobe has been carried out according to the usual techniques, either in a glass flask of 5 liters capacity, or in a stirred fermenter of 50 liters capacity, or again on a solid medium.
The bacterium was cultivated on different media: ordinary broth, ordinary gelose, ordinary broth enriched with Bactopeptone and with yeast extract.
The characteristics of the bacterium isolated are those of an aerobic of the genus Corynebacterium, namely:
Morphology: slender rod-shaped bacilli, rare forms of V-oriented club shapes, or in heaps, or in comb shapes. Gram +, having metachromatic corpuscles PA0 Culture: PA0 Energizing metabolism: PA0 Biochemical characteristics:
(a) On ordinary broth, clouds, then deposition and formation of a fog at the surface after some days; PA1 (b) On ordinary gelose: opaque colonies appearing in 24 hours, widening further assuming a yellowish color. PA1 Nitrate + (NO.sub.3 -NO.sub.2 +1) PA1 Urease + PA1 Catalase + PA1 Methyl red + PA1 Dextrin: 1.2.+-.3 PA1 Glucose + PA1 Levulose +
This germ, which has not been identifiable with any known species, has been denoted by the name Corynebacterium catarrhalis by Applicants and has been deposited in the National Collection of Microorganism Cultures held by the PASTEUR INSTITUTE, on the date Feb. 21, 1980, under the number I-116.